重组鸡β-防御素5-β-防御素10双分子蛋白的构建及其理化特性分析  

Construction of Recombinant Chicken AvBD5-AvBD10 Double Molecule Protein and Analysis of Its Physical and Chemistry Characterizations

作　　者：马得莹[1] 刘胜旺[2] 韩宗玺[2] 单安山[1] 

机构地区：[1]东北农业大学动物营养研究所,黑龙江哈尔滨150030 [2]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室禽传染病研究室,黑龙江哈尔滨150001

出　　处：《农业生物技术学报》2009年第1期41-46,共6页Journal of Agricultural Biotechnology

基　　金：诺韦司国际科学研究基金;黑龙江省教育厅科研重点项目(No.1153lz05);国家自然科学基金(No.30600435);国家重点基础研究发展规划(973)项目(No.2004CB117505)资助

摘　　要：鸡抗菌肽属禽β-防御素(AvBD)类,是鸡先天性免疫的重要组成部分。研究将AvBD10基因定向插入到AvBD5-pGEX SalⅠ和NotⅠ双酶切位点上,构建了AvBD5-pGEX-AvBD10双基因共表达重组载体。将重组质粒转化大肠杆菌(Escherichia coli)BL21,于37℃不同时间进行诱导表达,SDS-PAGE检测外源基因的表达。结果表明,重组AvBD5-AvBD10双分子融合蛋白的分子量约为36 kD,重组双分子蛋白占菌体总蛋白的35%,重组菌表达产物以包涵体形式存在。重组双分子蛋白经纯化后,分别以对数生长中期的大肠杆菌[BL21(DE3-)株]与致病性链球菌[Streptococcus(CAB株)]为检测菌,利用薄层平皿琼脂糖孔穴扩散法测定了重组双分子蛋白的抗菌活性,结果表明,重组双分子蛋白对这两种细菌都具有抗菌活性。并且对温度和pH有很高的稳定性,在-70～100℃或pH 3～12处理30 min仍具有抗菌活性。Chicken antimicrobial peptides, belong to β-defensins, are essential components of innate immunity and play significant roles in innate immunity in chickens. The cDNA of chicken avian β-defensins 10（AvBD10） gene was sub-cloned into Sal I and Not I sites of AvBD5-pGEX-6p-1 vector to construct recombinant plasmid AvBD5-pGEX-AvBD10. The recombinant plasmid was transferred into Escherichia coli BL21. Then the bacteria was induced with IPTG at 37℃ and different time, and exogenous expression was detected by SDS-PAGE. Results showed that a 36 kD protein which was equal to chicken AvBD5-AvBD10 protein in molecular weight was expressed in E.coli BL21. The production of AvBD5-AvBD 10 accounted for approximately 35% of the total protein. The recombinant fusion protein was purified and expressed as insoluble body. Two bacteria, E.coli and pathogenic Streptococcus of mid-log （10^8 cfu/mL） were used to evaluate antibacterial activity of the recombinant fusion protein by inhibition zone assaying. The results showed that the recombinant fusion protein exhibited anti-E.coli, and anti-pathogenic Streptococcus activity, and had high stability at various temperatures -70℃-100℃ and pH 3- 12.

关 键 词：鸡β-防御素 重组双分子蛋白 抗菌活性 

分 类 号：S188[农业科学—农业基础科学]